Colon cancer is the second most common cancer of men and women and the third leading cause of cancer mortality (1). It is estimated that $8.4 billion per year is spent caring for patients with this disease in the United States. Identifying chemopreventive agents and dietary constituents that facilitate the turnover of intestinal epithelial cells containing DNA damage and/or that block the progression of premalignant intestinal tissue to frank carcinoma could have a significant impact on public health. Similarly, novel agents that could be used to treat the 50% of colon cancer patients suffering from advanced or refractory disease are needed.
In mammalian cells, sphingolipid structure, composition and metabolism have been well characterized (2, 3). Knowledge of sphingolipid structure has facilitated in-depth analyses of the contribution of sphingolipids to membrane organization and their function in signal transduction events and normal physiology. Such studies have defined an important role for higher order sphingolipids in the formation of membrane subdomains (lipid rafts) wherein growth factor signaling and recruitment occur (4-7), and sphingolipid metabolites such as sphingosine, ceramide, sphingosine-1-phosphate and ceramide-1-phosphate have been shown to participate in signaling pathways regulating the key processes of cellular proliferation, migration, stress responses, apoptosis, angiogenesis and immune cell trafficking (8-13). Dysregulation of sphingolipid metabolism has been implicated as a contributing factor in carcinogenesis, cancer progression and drug resistance (14, 15). Recently it was shown that sphingolipid metabolism is disrupted in the mucosal epithelium during intestinal tumorigenesis and contributes to tumor progression (16, 17). In contrast, the accumulation of endogenous growth-inhibitory sphingolipids such as ceramide is recognized as a mechanism of action of some cytotoxic agents (18). The presence of the C4-5 double bond in the sphingoid base is believed to confer cytotoxic biological activity, as dihydrosphingosine and dihydroceramide are not cytotoxic in most cells. Strategies to enhance ceramide production and accumulation in malignant cells have been considered a novel approach for cancer therapy (19-21).
Sphingolipids are conserved throughout evolution and as such are enriched in many constituents of the human diet, including meat, dairy and soy products (22-24). Normal consumption of sphingolipids is estimated to be about 0.3-0.4 grams per day. Dietary sphingolipids are metabolized to ceramide and ultimately to sphingosine by brush border enzymes in the gut epithelium (25). Sphingosine (but not ceramide) is taken up by intestinal epithelial cells, where it inhibits growth (26, 27). Thus, intestinal epithelial cells are exposed to sphingolipids from both intracellular synthesis and from the diet. Dietary sphingolipids have been shown to be cytotoxic to colon cancer cells in vitro, and consumption of sphingolipids suppresses colon carcinogenesis in rodents (26-32).
Studies have been conducted on the physiological roles of sphingolipids in the genetically tractable organism, Drosophila melanogaster. In this species, tight regulation of sphingolipid levels is required for proper development, reproduction and the maintenance of tissue integrity, as demonstrated by the severe phenotypes observed in mutants with disrupted sphingolipid metabolism (33-37). However, a clear understanding of the role of sphingolipid metabolism and, in particular, the mechanisms by which sphingolipid metabolites influence physiological processes in this organism, has been hampered by an incomplete knowledge of the chemical structure of endogenous Drosophila sphingolipids and their metabolic products.
The AKT/PI3 kinase pathway has emerged as a central signaling node responsible for adjusting the metabolic and biosynthetic operations of the cell in response to changing nutrient and environmental conditions (Woodgett, J. R. “Recent advances in the protein kinase B signaling pathway.” Curr Opin Cell Biol 17(2): 150-157, 2005.; Ruggero, D. and Sonenberg, N., “The Akt of translational control.” Oncogene 24(50): 7426-7434, 2005). Activation of the pathway facilitates cell cycle progression, inhibits apoptosis, and enhances nutrient uptake and protein synthesis, leading to increased cell growth and survival. The PI3 kinase/AKT pathway is normally activated by tyrosine kinase receptor ligation, which activates class I phosphoinositol-3 kinase (PI3K). PI3K phosphorylates phosphatidylinositol-4,5-bisphosphate (PIP2), generating phosphatidylinositol-3,4,5-trlsphosphate (PIP3), which facilitates membrane recruitment and activation of the protein kinase AKT by virtue of the latter's lipophilic pleckstrin homology domain (PHD). Activated AKT subsequently phosphorylates downstream targets controlling cell growth and survival. The tumor suppressor PTEN antagonizes AKT signaling by catalyzing the dephosphorylation of PIP3 to PIP2 (Ramaswamy, S. et al. Proc Natl Acad Sci USA 96: 2110-2115, 1999). Activating mutations in the PI3K/AKT pathway are commonly found in human malignancies, including colon cancer (Tohma. Y., et al., J Neuropathol Exp Neurol 57(7): 684-689, 1998; Carpten, J. D., et al., Nature 448(7152): 439-444, 2007; Vivanco, I. and Sawyers, C. L., Nat Rev Cancer 2(7): 489-501, 2002).
Akt is a central player in the regulation of cell metabolism, cell survival, motility, transcription, and cell cycle progression (see, e.g., Fayard et al., Journal of Cell Science. 2005 118:5675-78). The Akt subfamily comprises three mammalian isoforms, Akt1, Akt2, and Akt3, which are products of distinct genes and share a conserved structure that includes three functional domains: an N-terminal pleckstrin homology (PH) domain, a central kinase domain, and a C-terminal regulatory domain.
Akt is a downstream component in the phosphoinositide 3-kinase (PI3K) pathway. Akt is activated by numerous upstream PI3K regulatory components, including, for example, receptor tyrosine kinases, integrins, B and T cell receptors, cytokine receptors, G-protein coupled receptors, and other stimuli that induce the production of lipid phosphatidylinositol 3,4,5 triphosphates (PtdIns(3,4,5)P3) through PI3K. Certain studies report as well that Akt can be activated in a PI3K independent manner (see, e.g., Vanhaesebroeck and Alessi Blochem. J. 2000, 346:561-576).
PtdIns(3,4,5) triphosphates serve as plasma membrane docking sites for both Akt and its upstream activator PI3K-dependent kinase-1 (PDK1) by interacting with their PH domains. Akt interaction with PtdIns-3,4,5-P3 promotes Akt translocation from the cytoplasm to the plasma membrane, where Akt undergoes phosphorylation at two sites: Thr308 in the activation loop and Ser473 in the carboxy-terminal tail. PDK-1 phosphorylates Akt at Thr308, but the Ser473 kinase remains unknown. Phosphorylated Akt translocates from the plasma membrane to the cytosol or the nucleus, where it acts on its downstream substrates.
When properly regulated, activated Akt eventually undergoes dephosphorylation by phosphatases and returns to the inactive state. For example, the tumor suppressor phosphatase and tensin homology (PTEN) and the SH2-domain containing inositol polyphosphate 5-phosphatase (SHIP) Indirectly inhibit Akt activity by regulating PtdIns(3,4,5)P3. In addition, protein phosphatase 2A (PP2A) and PH domain leucine-repeat protein phospatase (PHLPPα) directly dephosphorylate Akt at Thr308 and/or Ser473.
Akt mediates many of the downstream events regulated by PI3K. Of particular interest to cancer therapy. Akt regulates the activity of numerous substrates associated with cell proliferation, including, for example, p27. Chk1, Yes-associated protein (YAP), p53, murine double minute (Mdm 2). IκB kinase (IKK), Par-4, caspase-9, the family of forkhead box (Fox) transcription factors, p70 S6 kinase pathway through mammalian target of rapamycin (mTor) or Raf1, Bad, glycogen synthase kinase-3 a/β (GSK-3a/β), glycogen synthase, the androgen receptor, cyclin D1, and p21. Most of these substrates comprise the minimal consensus sequence RxTxx(S/T), where x is any amino acid and S/T is the phosphorylation site. Generally, Akt downstream activity promotes cell survival, for example, by either positively regulating cellular proliferation pathways or by preventing apoptosis. Although constitutive Akt action alone is believed to be insufficient for tumorigenesis, Akt over-activity is implicated in many types of cancer.
Also relevant to cancer therapy, Akt plays a dual role in cell motility, enhancing motility in certain cells such as fibroblasts, and inhibiting motility in other cells such as epithelial cells. For example, Akt expression in fibrosarcoma and pancreatic cancer cells increases their invasion through Matrigel, and Akt expression can promote epithelial-mesenchymal transition, a process associated with tumor progression to invasive and metastatic carcinoma. In contrast, as one example of Akt inhibition of motility, the Akt1 isoform in particular limits the invasive migration breast cancer cells, most likely by stabilizing Mdm2, and thus accelerating the degradation of the NFAT transcriptional regulator (see, e.g., Toker et al, Cancer Res. 2006, 66:3963-66).
Akt is also a regulator of insulin signaling and glucose metabolism, and its activation through the PI3K pathway is essential for most insulin-Induced glucose and lipid metabolism such as glucose uptake, glycogen synthesis, suppression of glucose output, triglyceride synthesis, as well as insulin-induced mitogenesis (see, e.g., Asano et al., Biol Pharm Bull. 2007, 30:1610-1616). For example, GLUT-4 is the primary glucose transporter in skeletal muscle cells and adipocytes, and plays a role in insulin-mediated glucose disposal. Insulin activates Akt through the PI3K pathway, causing GLUT-4 translocation to the plasma membrane and an increase in basal glucose transport. Akt effects glucose metabolism in other ways, as PI3K and Akt inhibitors inhibit not only insulin induced glucose uptake, but inhibit glycogen synthesis as well.
Studies also implicate Akt in intracellular bacterial growth (see, e.g., Kuijl et al., Nature 2007, 450:725-730). The Salmonella typhimurium effector SopB activates Akt1, which promotes bacterial intracellular survival by controlling actin dynamics through PAK4 and by controlling phagosome-lysosome fusion through the AS160-RAB14 pathway. Akt1 inhibitors may counteract the bacterial manipulation of host signaling processes, controlling the intracellular growth of bacteria such as S. typhimurium and Mycobacterium luberculosis. 
The Wnt signaling pathway involves a large number of proteins that can regulate the production of Wnt signaling molecules, their interactions with receptors on target cells and the physiological responses of target cells that result from the exposure of cells to the extracellular Wnt ligands. The canonical Wnt pathway involves a series of cellular events that occur when Wnt proteins bind to cell-surface receptors of the Frizzled family, causing the receptors to activate Dishevelled family proteins and ultimately resulting in a change in the amount of β-catenin that reaches the nucleus. Dishevelled (DSH) is a key component of a membrane-associated Wnt receptor complex which, when activated by Wnt binding, inhibits a second complex of proteins that includes axin, GSK-3, and the protein APC. The axin/GSK-3/APC complex normally promotes the proteolytic degradation of the β-catenin intracellular signaling molecule. After this “β-catenin destruction complex” is inhibited, a pool of cytoplasmic β-catenin stabilizes, and some β-catenin is able to enter the nucleus and interact with TCF/LEF family transcription factors to promote specific gene expression (see Gordon M D and Nusse R. J Biol. Chem. 2006 Aug. 11; 281(32):22429-33: Hausmann G, et al. Nat Rev Mol Cell Biol. 2007 April; 8(4):331-6; Clevers, H, Cell 2006, 469 (2006); Nusse, R. Science 316, 988 (2007)).
Activation of the Wnt signaling pathway leads to β-catenin migration into the nucleus, where it interacts with and converts the T-cell factor (TCF) transcription factor from a repressor to an activator, inducing target genes that mediate proliferation, adhesion and migratory responses (Fuchs, S. Y. et al. Cell Cycle 4(11): 1522-1539, 2005). Mutations in the Adenomatous Polyposis Coli (APC) gene, a negative regulator of the Wnt pathway, are responsible for intestinal polyposis in ApcMin/+ (also called Min) mice and colon cancer in the human disease Familial Adenomatous Polyposis (FAP) (Kucherlapati, R., et al. Semin Cancer Biol 11(3): 219-225.,2001; Sansom, O. J., et al., Genes Dev 18(12): 1385-1390, 2004). A high incidence of mutations in APC or other components of the Wnt pathway has been identified in sporadic colon cancer, indicating the central role that Wnt signaling plays in intestinal tumorigenesis (Sancho, E., et al., Annu Rev Cell Dev Biol 20: 695-723, 2004). In fact, activation of the Wnt pathway is considered one of the first molecular changes contributing to colon carcinogenesis (Kinzler, K. W. and Vogelstein, B. Cell 87(2): 159-170., 1996).
Accordingly, there exists a need for improved understanding of sphingolipid pathways and intermediates and of their role in influencing signaling pathways such as the AKT/PI3 kinase pathway and the Wnt signalling pathway, and their roles in cellular physiology, including molecular mechanisms of cancer. Disclosed herein are compositions and methods that address this need by providing unsaturated sphingosine compounds that are useful in treating cancer and other diseases, and which offer other related advantages.